The present invention concerns a mass spectrometer combined with a sample separation apparatus used for separation and analysis of mixed biological samples, for example, sugar, peptide and protein.
In the field of analysis, an importance has been attached to the development of mass spectrometry for biological compounds at present. Since the biological compounds are usually dissolved as a mixture in a solution, development has been progressed to a mass spectrometer combined with the sample separation apparatus for separating the mixture. As a typical example, there can be mentioned a combined apparatus of capillary electrophoresis apparatus-mass spectrometer utilizing capillary electrophoresis for the separation of the sample. The capillary electrophoresis is excellent in the separation of the mixture but can not identify substances. On the other hand, the mass spectrometer has a high analyzing sensitivity and is excellent for the ability of identifying substances but analysis of the mixture is difficult. In view of the above, a sample is separated by the capillary electrophoresis apparatus and the separated sample is analyzed by the mass spectrometer. Thus, the mass spectrometer combined with the capillary electrophoresis apparatus is much effective for the analysis of a mixture.
An existent mass spectrometer combined with the capillary electrophoresis apparatus described above is described in Analytical Chemistry, Vol. 60, No. 18, Sep. 15, 1988, pp. 1948-1952. The existent mass spectrometer will be explained with reference to FIG. 13. In the mass spectrometer of the prior art, an electrospray ionization method is used for ionization of a sample. A capillary 1 is a fused-silica capillary having an outer diameter of about several hundreds micrometer and an inner diameter of about several tens micrometer. The inside of the capillary 1 is filled with a buffer solution. A sample solution is introduced from one end 2a to the inside of the capillary 1. After introduction of the sample solution, the end 2a is kept in a buffer vessel 4 filled with a buffer solution 3. The other end 2b of the capillary 1 is inserted to the inside of a metal tube 5. Generally, a flow rate of a buffer flowing through the capillary is small and it is often difficult to nebulize the sample solution stably and continuously. Then, a sheath liquid 6 is introduced in a gap between the capillary 1 and the metal tube 5 for assisting nebulization. When a high voltage is applied from a high voltage power source 7a between one end 2a of the capillary 1 and the metal tube 5, since the end 2b of the capillary 1 is electrically connected by way of the sheath liquid 6 with the metal tube 5, a high voltage is applied between both ends 2a and 2b of the capillary 1. Thus, the sample is sent to the end 2b while undergoing electrophoretic separation in the capillary 1.
The sample reaching the end 2b is mixed with the sheath liquid 6 and then electrosprayed by a voltage applied between the metal tube 5 and an opposing electrode 8a by power source 9 for a nebulizer. Ions relevant to the sample molecules are contained in droplets formed by the electrospray. The ions relevant to the sample molecules are entered through a sampling aperture 10a into a differential pumping region 12 evacuated by an evacuation system 11a and, further, enter a vacuum region 13 evacuated to a high vacuum degree by a vacuum system 11b. The ions entering the vacuum region 13 are subjected to mass separation in a mass analysis region 14 and the mass-separated ions are detected by an ion detector 15. A detection signal from the detector 15 is sent by way of a signal line 16 to a data processing apparatus 17 and put to data processing to obtain a result of mass spectrometry for the sample substance.
In the existent mass spectrometer combined with the capillary electrophoresis apparatus described above, electrospray ionization is used for ionization of the sample. The electrospray ionization is a method of taking out highly polar substances such as protein or peptide present as ions in a solution as gaseous ions. Therefore, neutral substances not possessing charges in the solution can not be detected at a high sensitivity in the mass spectrometer combined with the existent capillary electrophoretic apparatus. Since such neutral substances include, for example, amines in various kinds of medicines and neutrotransmitters, it is extremely important to analyze electrically neutral samples for the study in the field of biotechnology or medicine.
Further, as one of methods for separation of samples by capillary electrophoresis, micellar electrokinetic chromatography has been known. In the micellar electrokinetic chromatography, micelles are formed by adding a surfactant to a buffer solution, and a neutral substance not having charges is separated by utilizing the difference of distribution when each of the sample compounds is distributed in the micelles. Also in this case, for extending an application range of the mass spectrometer combined with the capillary electrophoresis apparatus, it has been desired for the development of an apparatus capable of analyzing, at a high sensitivity, neutral substances having no charges in the solution.
Further, the ion intensity obtained by the existent electrospray ionization method is approximately given by the following equation Electrophoresis, Vol. 14, 1993, pp. 448-457:
I(A+)xe2x88x9dV(A+)/V(C+)xe2x80x83xe2x80x83(1)
where I(A+) represents a signal intensity of ion A+ as an object of analysis, V(A+) represents a flow rate of ion A+ to be analyzed, and V(C+) represents a flow rate of contaminant ions other than ion A+ to be analyzed. Accordingly, for attaining mass spectrometry at a high sensitivity by using the electrospray ionization method, it is important to remove contaminant ion C+ in the sample solution.
On the other hand, in the capillary electrophoresis method, a method of adding a salt at high concentration in a buffer solution for electrophoresis is generally used for preventing sample molecules from adsorbing on wall surfaces or the like. Accordingly, since contaminant ions (for example, Na+, K+) formed by dissociation of the salt are contained in a great amount in the ions obtained by electrospray, the denominator: V(C+) in the formula increases remarkably to reduce the signal intensity of the ion as an object of the analysis. Accordingly, in the existent mass spectrometer employing electrospray for the ionization of the sample, it was difficult to obtain a signal of the ion as an object of analysis at a sufficient intensity.
Further, in micellar electrokinetic chromatography, analysis is effected by forming micelles of a surface active agent such as SDS (sodium dodecyl sulfate) in a buffer. For forming the micelles, it is necessary to add a surfactant at a concentration exceeding a critical value (critical micelle concentration) in the buffer. Under micelle-forming conditions, cations and anions liberated from the surfactant are present in a great amount as contaminant ions in the buffer. Therefore, in the existent apparatus using the electrospray ionization method, measurement of the sample molecular ions is difficult by the effect of the contaminant ions.
With the reasons described above, it has been strongly demanded for providing a mass spectrometer combined with a sample separation apparatus such as a capillary electrophoresis apparatus improved so as to less undergo the effect of the salt in the buffer.
A first object of the present invention is to provide a mass spectrometer capable of separating an electrically neutral substance present in a solvent which was difficult to be ionized by an existent electrospray ionization method and analyzing the same at a high sensitivity.
A second object of the present invention is to provide a mass spectrometer capable of using, to a sample separation apparatus, a buffer for electrophoresis which was difficult to be used in an existent mass spectrometer combined with a capillary electrophoresis apparatus.
In accordance with the present invention, a sample solution is separated by using a sample separation apparatus such as a capillary electrophoresis apparatus, the separated sample solution is nebulized by flowing from a capillary, gaseous sample molecules formed by vaporization of liquid droplets resulting from nebulization are ionized by chemical reaction, and the ions of the thus obtained sample molecules are subjected to mass spectrometry in a mass analysis region. The nebulization, vaporization and ionization are conducted in an air under an atmospheric pressure or a reduced pressure.
FIG. 1 shows a basic constitution of a mass spectrometer according to the present invention by using a capillary electrophoresis apparatus as a sample separation apparatus. In FIG. 1, a sample separated in a capillary electrophoresis region 18 is nebulized together with a buffer solution in a nebulization region 19. Liquid droplets formed by nebulization are vaporized in a vaporization region 20. Gaseous sample molecules formed in the vaporization region 20 are ionized in a chemical ionization region 21 by chemically reacting with ions derived from gaseous molecules present in the ionization region 21. For promoting the ionization by the chemical reaction, a corona discharging process to be described later may be used.
Ions relevant to the sample molecules obtained in the ionization region 21 enter by way of a sampling aperture 10a into a differential pumping region 12 evacuated by a vacuum system 11a and, further, enters passing through a sampling aperture 10b into a vacuum region 13 evacuated to a high vacuum degree by a vacuum system 11b. Ions entering the vacuum region 13 are put to mass separation in a mass analysis region 14 and detected by an ion detector 15. A detection signal from the ion detector 15 is sent by way of a signal line 16 to a data processing unit 17 for data processing.
The chemical ionization region 21 may be disposed in the differential pumping region 12. The inside of the differential pumping region 12 is kept at a pressure from several Pa to several hundred Pa. Accordingly, the sample molecules collide against gaseous molecule ions present in the differential pumping region to form ions of the sample molecules by the chemical reaction.
As the separation mode in the capillary electrophoresis region 18, there can be mentioned various modes such as capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing electrophoresis and micellar electrokinetic chromatography. In the capillary zone electrophoresis, a free solvent is filled in the capillary and the sample is separated due to the difference of the mobility of the sample. In the capillary gel electrophoresis, a gel is filled in the capillary and the specimen is separated by utilizing the molecular sieve effect of the gel. In the capillary iso-electric focusing electrophoresis, a gradient is provided to a hydrogen ion concentration in the capillary and the sample is separated depending on the difference of isoelectric point of the sample. In the micellar electrokinetic chromatography, micelles formed by adding a surface active agent to the buffer solution, and the sample is separated by utilizing the difference of distribution of the micelles to each of the sample compounds. In the present invention any of the separation modes described previously may be used.
In the nebulization region 19, the sample solution can be nebulized by using a nebulizing means using an electrospray means, nebulization by heating, pneumatic nebulization means or nebulization means using ultrasonic oscillator. In the vaporization region 20, the nebulized sample solution can be vaporized by using vaporization means such as a heated metal block or infrared irradiation.
In the chemical ionization region 21, ions relevant to sample molecules A are formed mainly by the following proton addition reaction or proton elimination reaction assuming the sample molecule as an object of analysis as A and gaseous molecules chemically reacting therewith as B:
A+BH+xe2x86x92AH++B (proton addition reaction)xe2x80x83xe2x80x83(2)
A+Bxe2x88x92xe2x86x92(Axe2x88x92H)xe2x88x92+BH (proton elimination reaction)xe2x80x83xe2x80x83(3)
For instance, hydronium ion (H3O+) or cluster ion thereof [H3O+(H2O)n] are formed by generating corona discharge in atmospheric air. The thus formed ions react with the sample molecules A as shown below to form ions AH+ relevant to the sample molecule A:
A+H3O+xe2x86x92AH++H2Oxe2x80x83xe2x80x83(4)
A+H3O+(H2O)nxe2x86x92AH++(n+1)H2Oxe2x80x83xe2x80x83(5)
In this way, when the sample solution reaching the exit end of the capillary is nebulized and the resultant gaseous sample molecules are ionized by the chemical reaction, ions relevant to the sample molecules not having charges in the solution can be obtained. When the thus obtained ions are subjected to mass analysis in the mass analysis region, sample molecules having no charges in the solution can be analyzed. As a result, the application range of the mass spectrometer combined with the capillary electrophoresis apparatus can be extended remarkably.
Further, in an existent mass spectrometer using the electrospray ionization method, ionic substances ionized in the solution can also be detected at a high sensitivity. On the other hand, in the present invention using the chemical ionization method by corona discharge, such ionizing substances are less detected rather. This is probably attributable to that since the ionic substances flies as gaseous ions toward the sampling aperture 10a merely by being nebulized (electrosprayed) in the nebulization region 19, the flying trace is bent by an electric field for generating corona discharge in the ionization region 21 and can not reach as far as the sampling aperture. That is, the sample molecules carrying no static charges and reaching as far as the ionization region 21 is at first ionized and analyzed by the chemical ionization method in the ionization region 21. Namely, the sample molecules that can be analyzed in the mass spectrometer according to the present invention are mainly neutral molecules in the solution, whereas the sample molecules that can be analyzed in the existent mass spectrometer are mainly ionic molecules in the solution. As described above, the mass spectrometer according to the present invention and the existent mass spectrometer have a so-called relationship complementary to each other. The mass spectrometer according to the present invention combined with the capillary electrophoresis apparatus has a low sensitivity to ions derived from a salt if it is incorporated in a buffer for electrophoresis. In addition, the range for the selection of the buffer solution can be extended in the mass spectrometer according to the present invention, compared with the existent mass spectrometer combined with the capillary electrophoresis apparatus. Accordingly, the application range of the mass spectrometer combined with the sample separation apparatus such as the capillary electrophoresis apparatus can be extended outstandingly according to the present invention. As the sample separation apparatus, liquid chromatographic apparatus can be used in addition to the capillary electrophoresis apparatus described above. Further, if separation of the sample solution is not necessary, the sample solution may be introduced by a flow injection method into the capillary and then nebulized from the exit of the capillary.
These and other objects and many of the attendant advantages of the invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings.